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HPLC

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HPLC

originally referred to:

High Pressure Liquid Chromatography

high pressure to be able to use small particle size to allowproper separation at reasonable flow rates

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PRINCIPLE

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Principle of HPLC:The principle of HPLC are based on Van Deemter equation which

relates the efficiency of the chromatographic column to theparticle size of the column, molecular diffusion and thickness ofstationary phase.

The Van Deemter Equation is given as

H or HETP = A + B + C

where, A represents eddy diffusion

B represents molecular diffusionC represents rate of mass transfer

represents flow rate

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Basic Components of an HPLC

System1.Pump System.Mobile phase pressures up to 6000 psi are necessary to

achieve reasonable column elution times (~ minutes). Typical flowrates are 0.1 to 10 mL/minute.

2.Injection System.Used to introduce small samples (0.1 to 500 L) intothe carrier stream under high pressure.

3.Reservoirs (Solvents).Multiple solvents are necessary for performinggradient elution's (i.e. changing the polarity of the mobile phaseduring a run).

4.Chromatographic Column. Typically 10-30 cm in length containing apacking of 5-10 m diameter. Many types of columns are available,depending on the type of liquid chromatography desired.

5.Detector.Many types are available including UV, IR, refractive index,fluorescence, conductivity, mass spectrometry, and electrochemical.Diode array detectors are used when wavelength scans are desired.

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HPLC Basic Instrumentation

Mobile

phasePump

Solvent Delivery

Injector

Sample Injection

Column

Separation

Detector

Data Processor

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HPLC system

Solvent Reservoir

Degasser

Solvent Delivery System (Pump)

Injector

Column &oven

Detectors

Recorder (Data Collection)

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PUMP SYSTEM

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HPLC PumpHPLC Pump

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Solvent Reservoir

Usually one or more glass or stainless steel reservoirs each

of which contains 200-1000 ml of solvent

Isocratic elution - single solvent separation technique Gradient elution - 2 or more solvents, varied during

separation

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SAMPLE INJECTION SYSTEM

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HPLC Autosampler and InjectorHPLC Autosampler and Injector

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from Pump from Pump to Column

Vial

Needle

Measuring Pump

to Column

LOADLOAD INJECTINJECT

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HPLC ColumnHPLC Column

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HPLC DetectorHPLC Detector

UV/Visible SpectrophotometerUV/Visible Spectrophotometer

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Column

straight, 15 to 150cm in length; 2 to 3mm i.d.

packing - silica gel,alumina, Celite

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CHARACTERISTICS OD

DETECTORS Have high sensitivity and the same predictable response Respond to all solutes, or else have predictable specificity Have a wide range of linearity Be unaffected by changes in temperature and mobile-phase

flow

Respond independently of the mobile phase Not contribute to extra column band broadening Be reliable and convenient to use Have a response that increases linearly with the amount of

solute Be nondestructive of the solute Provide qualitative information on the detected peak Have a fast response

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HPLC

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Degasser

Problems caused by dissolved air(O2, N2)in mobile phase

Unstable delivery in pump

Bigger noise and large baseline-drift in detector cell

In order to avoid causing the problems,

mobile phase should be degassed.

vacuum pumping systems

distillation system

a system for heating and stirring the solvents

sparging system - bubbles an inert gas of low solubility through the

solvent

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Solvent Delivery System

Requirements

ability to mix solvents and vary polarity of mobile phase during

run

unlimited solvent reservoir

generation of pressures up to 6000 psiflow rates ranging from 0.1 to 10 mL/min

flow reproducibilitys of 0.5 % or better

resistance to corrosion by a variety of solvents

pulse-free output

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Solvent Delivery System

RheodyneInjector

%A %B %C Flow Rate Pressure

{H2O} {MeOH} (mL/min) (atmos.)

Ready

Ternary Pump

A

C

B

from solventreservoir

Co

lum

n

todetector

to column

through

pulse

dampener

to injector

through pump

load

inject

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Chromatography II: HPLCHPLC Waste CollectionHPLC Waste Collection

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Use a 10 mL volumetric pipet to add 10.00 mL of soft

drink to 10.00 mL of deionized water.Mix thoroughly and half fill a HPLC vial with

your sample. Label the vial with your name and thename of the soft drink.

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Analysis of ResultsUse the four caffeine standards to prepare a

calibration curve (graph) that plots peak area vs.

concentration.

Draw a best fit straight line on your graph.Use your calibration curve to determine the

concentration of caffeine in the sample you prepared.

Use the concentration of caffeine in your sample,

along with the dilution equation, to determine theconcentration of caffeine in the soft drink you used.

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HPLC Chromatogram of Standard 1HPLC Chromatogram of Standard 1

0.500 x 100.500 x 10-4-4 M caffeineM caffeine

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HPLC Chromatogram of Standard 2HPLC Chromatogram of Standard 2

1.00 x 101.00 x 10-4-4 M caffeineM caffeine

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Calculations

Remember, the original soft drink was diluted to prepare your HPLCsample. The concentration (M) of caffeine in the original soft drink can

be calculated by using the dilution equation:M1V1 = M2V2

V2

M1

V1M2 =

The concentration (mg caffeine / 500 mL soda) of caffeine in a full can

of your soft drink can be determine using the following equation:

M2 x 194.2 g caffeine x 1000 mg x 0.500 L = mg caffeine

mol caffeine g in bottle

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HPLC Applications

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HPLC - Applications

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